Science topic
Neuroimmunology - Science topic
Neuroimmunology are neuroscience, Immunology,Neuroimmunological interactions during physiological conditions and disease
Questions related to Neuroimmunology
If you are aware of the fact that cancer patients treated with standard chemotherapy over the past 20 or so years have cognitive problems with reasoning and motor skills, I'd like your input on why, and why ten to twenty years later. Microglia (brain macrophages, which is a misnomer) can initiate the process of neurodegeneration, which leads to ex-cancer patients, who've survived through chemotherapy, down the path of significant brain damage. There is an initial insult due to systemic inflammation, but I'm thinking that endothealial damage and their ability to secrete inflammatory lymphokines perpetuate or exhasterbate chemotherapy-induced cognitive problems in cancer patients.
What do you think?
Michael
I am analyzing the immune cells in neuronal cell bodies using flow cytometry and noticed two diagonal populations in my FSC-A vs. FSC-H plot. I have excluded the dead cells.
Is it possible that both populations are singlets?
I've attached the plot below.

Hi, I'm a student who studies neuroimmunology.
I want to analyze mouse vagus nerve with RT-qPCR like this thesis but I don't know how to dissect the vagus nerve I found some protocol videos for DRG neurons from a mouse but there are not many protocols for dissecting VG, and even there are no protocol video for VG nerve dissection in Youtube. Could you recommend any good protocol for VG dissection?
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RNA isolation from mouse DRG and JG/NG (VG) and qRT-PCR
Mouse DRG or nodose ganglia (NG)/jugular ganglia (JG) (combined VG) were harvested and homogenized with a bead homogenizer (BioSpec) in lysis buffer RA1 (Macherey-Nagel). Total RNA was extracted with the NucleoSpin RNA isolation kit following manufacturer’s protocol. Equal amounts of cDNA were synthesized from total RNA extracts using the iScript cDNA Synthesis kit. Gene expression levels were determined using gene-specific primers (key resources table) and 2X qPCR Universal Green MasterMix (Lamda Biotech) or Power SYBR™ Green PCR Master Mix (Thermo Fisher Scientific). Reactions were cycled using a StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific) or QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher Scientific) using the manufacturer’s protocol. Gene expression was normalized to Gapdh, and relative expression was calculated using the ΔΔCt method.
Hello! I've recently been trying to optimise immunofluorescent staining of mouse meningeal tissue, and I'm having trouble mounting it just because it's so thick.
We use prolong to mount and usually ~10 uL is enough for our 10 uM brain cryosections but even 20 uL seemed to be too little for the meninges since the meninges is much thicker (cover slip wouldn't rest on the tissue properly).
Would anyone have any advice on this? I feel like the process would be similar to mounting thicker cryosections of regular tissue.
I was wondering if anyone knows an antibody that can be used for staining microglia in zebrafish? I am familiar with 4c4 and L-plastin antibodies, but these are not perfect for me. Is anyone familair with, for example, a P2Y12 ab that works in zebrafish ?
Thanks!
I always hear about some myths in neurology, and mostly about stroke but I want to know what myths other diseases have.
A peer-reviewed journal, Neuroimmunology and Neuroinflammation opened a special issue to publish review articles about recent advances on Parkinson's disease. Please refer the link above to have more information how to submit it. Thanks.
Backil
Hello experts,
I want to ask about CNS in the Neuroinflammation/Neuroimmunology concept.
Is it correct to say that CNS is defined as immune privilege organ due to its unique of immune reaction compare with the general immune system? Is the unique refers to the ability of CNS to induce its own immune reaction, independent from other peripheral immunity, or any other reasons?
And is it still updated to classify CNS as immune privileged system?
Thanks in advance for your answers and comments
Hello everyone,
I was wondering if anyone knows another protocol for myelin purification other than the one published by Norton & Poduslo, 1973.
I want to do a phagocytosis assay ex vivo with primary microglia and myelin, and in vivo I want to inject it into the prefrontal cortex of mice to evaluate phagocytosis activity of the microglia from that region.
However, I'm finding a bit difficult to get access to an ultracentrifuge as described in the Norton & Poduslo method.
Any suggestions are well appreciated.
thank you all
Hello everybody
I've been trying to seed microglia from adult mice and I haven't been successful.
I isolated them using Miltenyi Beads and that worked perfectly, passed them through the cytometer and confirmed my isolation worked good and about 85% were viable. I seeded them in 24-well plates with Poly-L-Lysine but microglia didn't make it throughout the night.
Any suggestions?
Thank you
Over the past three years, a patient with hereditary desminopathy (Thr341Pro DES mutation in the heterozygous state) has an uneven decrease in muscle strength in the hands. In the right hand, the decrease in muscular strength in the last three years is 2.1 times, and in the left one - 1.5 times. In a weaker limb, the disease progresses faster. A similar pattern is observed in the legs. The father of the patient had the same changes.
Hi guys!
I am advancing in the empirical design of my next project about regimes of knowledge production on Adverse Drug Reactions (ADRs) and I would like to know if you have any suggestions of literature about this topic in STS, Medical Sociology of related fields.
I am interested in the perspective of the coproduction of biomedical evidence which emerges in the circuit of pathologization/diagnostic/medicalization/evaluation/new research agenda, specially addressed to the diffusion of immunotherapies and artificial antibodies in Oncology and Rheumatology.
Keep safe and cheers!
Renan
Within three years, a patient with a desminopathy (Thr341Pro DES mutation) was found to have a 17% increase in the level of C4 complement components to 0.41 g / l (Norm 0.1-0.4 g / l).
Does OLig2 ab stain all oligodendrocytes? i.e. progenitor, postmitotic and mature oligodendrocytes in embryonic , postnatal and adult brain. I mean is there a single antibody that can stain all developmental and mature oligodendrocyte (regardless the developmental stage).
If not, I would appreciate if anyone recommend a master marker for all oligodendrocytes.
I want to look at the expression of CD68 (ED 1)- marker of activated microglia in rat (sprague dawley) brain sections (hippocampus and medial prefronal cortex). The animals were perfused transcardially with saline followed by 4% PFA. 40um sections were then cut on a vibratome. I tried the abcam anti CD68 antibody (ab 31630) with different antigen retrieval methods (boiling, boiling+citrate and boiling+Tris) but could not detect any staining. I had also stained the sections simultaneously for Iba-1, a general marker for microglia which worked excellently. I want to know:
1) If anyone knows of a CD68 antibody which works well in rat brain sections and the protocol for the IHC
2) If anyone has successfully been able to detect CD68 expression using the abcam antibody. If yes, then kindly share the protocol.
Thanks!
I want to study the demyelination degree of EAE mice. I know that in the EAE model, lesions are preferentially located in the spinal cord and due to that it is very common to use LFB staining for spinal cord. Nevertheless I´m also interested in analyzing the brain. Has anyone used this staining in brain frozen tissue?
Thank you very much in advice
Basically, I am comparing gene expression in different neuroinflammatory pathways using mRNA Seq in a transgenic (KO) mouse model. The main goal is too see what genes are compensating during a neuroinflammatroy response.
What other experiments often accompany an RNA seq focused project? Western Blot? IHC? qPCR validation? And what would the purpose of those experiments serve in further elucidating the role of this missing gene and the henceforth compensatory mechanisms?
This is for my Masters thesis project so any advice would be helpful!
i can not find any foundations supporting cancer immune therapy and cancer neuroimmunology research in egypt or outside. and if there how can i contact with them?
I have been recently trying to isolate primary microglia from mice pups brain (P0-P1). To prepare mixed microglia culture, I used 3 brains for a 75 cm2 flask. After around 10-12 days, astrocyte confluent layer is formed, and many microglia appears. I isolated those microglia by a shaking method (100-120 rpm/ 2 hrs).
At first harvest, I usually get 3-5 x 105 cells per flask. But, after the first harvest, I do not see the growth of new microglia. I only see the microglia that did not detach during the first harvest. Nevertheless, I tried to isolate for second harvest (after 1-2 weeks from 1st harvest), but end up with very low yield (2 x 104 cells).
Is there any ways to increase the yield?
Medium used: DMEM (10% FBS, 1% penicillin/streptomycin)
Over the past four years, in a patient with desminopathy (Thr341Pro DES mutation in the heterozygous state), the majority of antioxidant status indicators increased 1.2-2.0 times. Including glutathione in the blood increased 1.8 times to a value of 896 μmol / l (the norm of 500 - 1500 μmol / l), and the level of coenzyme Q10 in the blood increased 1.8 times to a value of 0.8 mg / l (norm 0,4 - 1,6 mg / l), vitamins E and C increased by 1.3 times, respectively, to 6.4 and 6.2 μg / ml.
In the literature there are conflicting data on their effect on the human body.
Our neuroimmunology lab currently has a BD Accuri C6 that has broken about every year and a half since it’s purchase (about 5 yrs old). Valve issues, fluidics leaking onto electronics, flow cell issues, laser problems, etc. We have about 50k to spend on a new flow cytometer. BD has said they’ve fixed these issues, but I am hesitant. We only need the two lasers and 4 color system. We mostly stain immune cells with FITC, PE, PerCPCy5.5 (or 7AAD), and APC panels. It’s used about every other day for an hour or two. I’ve been researching the NovoCyte, Guava easyCyte, and CytoFLEX too, but my PI wants to stick with the familiar (plus they are running a great deal at about 38k with 3 years warranty). Anyone have experience with the new BD Accuri C6 Plus?
Over the past six months, a patient with hereditary desminopathy (Thr341Pro DES mutation in the heterozygous state) has a 1.6-fold increase in the level of the pro-inflammatory cytokine interleukin-6 to a value of 8.77 pg / ml (<7.00 pg / ml).
In addition, several parameters indicate the presence of inflammation: an increase in the number of immunity cells with markers CD25 +, CD95 +, HLA-DR + in the T-cell link; as well as an elevated level of C4 complement components.
The level of other cytokines Il-1β, Il-8, Il-10, TNF-α is normal.
At the same time, the immunoregulatory index fell below the norm and is 1.19.
Details are indicated in the questions of this project "myofibrillar myopathy".
Dear scientists, please, join the discussion on this issue, do not limit yourself to viewing only. Ask me additional questions, send messages to your personal mail. Ready to answer any questions and listen to wishes.
Some tips, protocols, etc will be gratefully accepted for non paraffinized tissues but cryosections of spinal cord of 18um thickness.
I am planning to design and synthesis of novel small inhibitors/antagonist of TLRs.
How should I begin? I have requested many private companies to provide samples but they could'nt provide as per their policies. So please help in this regard.
The chemistry of the Kit is based on two classical chemical reactions: Griess and Saville.
Is there any data comparing cyclophosphamide and MMF in acute neurolupus?
how much Brdu should be injected in mice to address smooth muscle proliferation like DSS induced Colitis?
I am attempting to label proliferative cells in the rat brain (Hippocampus) using BrDU intraperitoneally. However, we are able to detect BrDU in gut cells, but not in the brain. Does anyone know why?
I know there was debate as to weither once th2 always th2 while some thought there could be switching between th1 side and th2 side, while looking at profiles with GWI/ME/CFS some showed th1/th17 while others showed th2 side. I'm WONDERING IF WE MIGHT SWITCH BACK AND FORTH, DEPENDING ON WHAT MAY BE GOING ON AT THE TIME, MAYBE TH1 POINTING TO EXPOSURE OR RE-EXPOSURE TO A TRIGGER WHILE TH2 MAY POINT AT ACTIVE INFECTION WEITHER IT'S A REACTIVATED INFECTION OR A NEW ONE.
I want to contruct a primer for assay of scavanger receptor marker MARCO , or CD163 for rat animal model. So my question is that how to consttruct a primer for this ?
Does anyone works with this molecule in ratt tissue? I am trying to find the best commercial antibody in order to detect CB1 receptor by immunofluoresce and western blot.
Hello everybody,
I am searching for Anti-Bradykinin Receptor B2 Antibody and the phosphorylated one. I will be using brain sections from Guinea pigs and do the immunofluorescence stain.
Note: This is the first time for me to work with Guinea pigs, and its difficult to find antibodies compatible with it.
Thank You in advance.
Hello,
I am trying to optimize our Olig2 immunohistochemistry staining protocol for PFA fixed mouse brain. I found a very helpful website that reviewed several recent publications and found people used concentrations from 1:100 to 1:500 on mouse brain, and I am planning to try several of them (https://www.labome.com/review/gene/mouse/Olig2-antibody.html) . However, on the abcam website they advise to perform heat mediated antigen retrieval with citrate buffer pH 6 for this antibody. I was wondering if anyone tried antigen retrieval for Olig2 IHC?
Otherwise, if you have experience with antigen retrieval using a microwave, could you please advice the steps? I found a protocol suggesting placing tissue sections on slides, in citrate buffer (pH 6), and boiling for 20 minutes, whereas another protocol suggested boiling for 7 minutes.
I was also wondering, after antigen retrieval, what is the best way to continue the staining protocol having the sections already on slides: i.e. the incubation with the primary antibody over night etc? I have only ever stained with the tissue inside well plates, not on slides.
Thank you,
Carola
Does anyone know the brain concentration or % of antibody that was able to pass the BBB in patients treated with Abeta antibodies bapineuzumab or solanezumab? Thank you.
My antibody didn't work at all. I have mouse brains, 30 um cryosectioned, and tried different concentrations of tween, as well as both formamide and SSC treatment to no avail.
I need to label free-floating coronal mouse brain sections. Lamentably the typical SC-52 antibody of Santa Cruz Biotechnology has been discontinued. They suggested me to try with c-Fos (E-8): sc-166940 antibody but there are not many references about it.
Thanks very much.
Dear researchgaters,
I have a question regarding staining protocols for oxidative tissue markers via IHC (4-HNE, MDA-2, 8-0HdG etc). I have found that these markers work in cryo stored material, but I have not had any success getting them to work on paraffin material. When staining with cryo, due to the poor freezing method of the brain tissue, I often have poor quality stains, high background and poor morphology on some many aspects of cells or tissue. As the tissue for paraffin is superb in terms of quality and structure, it would be ideal to get these markers to work on this tissue.
For cryo staining I:
Acetone fix 10 min, air dry 10 min, PBS soak 10 minutes, antibody block (10%fcs + TBS or Dako) for 1/2 h -> Primary antibody overnight at 4c.
Some protocols I have seen where people stain parrafin material they are not using a retrieve step but are using .1% triton in their blocks. I have tried this during EDTA retrieve but no luck. I have tried Citrate/EDTA retrieve, and also proteinase K. Using the EDTA/Citrate retrieve I get very beautiful staining of p22phox, iNOS, SOD2, SOD1 on the parrafin embedded tissue. Can someone please help me out here!
Hello!
I am having trouble deciphering between fluorescent-tagged latex beads and E.coli particles that are just bound to the macrophages cell surface vs. engulfed.
Has anyone had success washing off surface-bound latex beads or bacteria?
I am currently growing my cells on coverslips, incubating with the beads/e. coli, followed by washing with PBS 3X. However, there are still many beads bound to the glass coverslip after washing. I would like to isolate only the engulfed beads!
CD3, CD5 Parrafin section immunohistology.
Dear Colleagues, I have also problem in Immunostaining of Parrafin sections of Rat sciatic nerve and spleen with CD3 and CD5. I am also using rabbit anti-human CD3e, #A0452, Dako as primary antibody and using Heat method for antigen retrival, but still i am unable to establish T cell staining. Please can any body gives some suggestion to improvise my stainings. Thanks
I have already perfused rat and fixed the brain and contusion spinal cord tissue with 4% PFA . Gonna use them for immunohistology. But now I don't have time to dehydrate and embed them into paraffin blocks immediately. I would like to know in which buffer it is convenient to leave the samples, if I want to include them in paraffin about a month (for example, ethanol, PB) or for a long time?
I am doing double immunohistochemistry staining of rat brain sections. Unlike most protocols, I work with thicker sections (40μm, it still works very well) with all incubations done in a tissue culture plate. I follow the same protocol every time, I mix the sections during all incubation steps and still I get different staining intensities every time! Sometimes even the two hemispheres of a single section are coloured differently! I tried to optimize the conditions in multiple ways - I tried 12-well, instead of 24-well plate, with higher volumes, more mixing, extended incubation times, less sections per well, etc., yet this doesn't improve enough, as I expected. I still can't achieve regular staining and more importantly - easily comparable between experiments! Even the final step - the substrate reaction - develops differently every time! Also, the background level is hard to assess. I guess I'm missing something, I will be glad if someone with more experience can give me a clue, or has encountered similar issues, to share some suggestions on how to improve.
I am staining for c-Fos and calbindin, biotinylated secondary antibodies + streptavidin-HRP. I use two different substrates - standard DAB and a two-component Vector SG peroxidase substrate kit. All concentrations should even be in surplus. Tissue preparation has been performed similarly from the start.
Thank you!
I would like to perform immunoprecipitation with an anti-v5 antibody followed by a mass spect analyses, on brain extract tissue.
Where may I find the most suitable protocol ? ( anti-v5 references, which beads or which buffer should I use, and which quality control I should do )? Many thanks in advance for your answers :).
Marie.
I am trying to use different antibodies working for trasmission electron microscope (TEM) to mark satellite glial cells (SGCs) in mice Dorsal Root Ganglia (DRGs) but none seems to work. Does anybody ever try it?
I did couple of stainings with Egr-1 in mice and I always end up with a lot of background which makes it very difficult to differentiate the positive nuclei in the tissue. I would appreciate any experience with this
Hello. Could anyone, please, help me? I have made immunofluorescent staining ща 3 DIV primary neuronal culture made from P1 mice hippocampus. How should the beta-3 Tubulin looks like on 3 DIV? I have attached an image - can it looks like this or this is a nonsense?
Thank you.
I have read papers highlighting ZO-1's sensitivity to overfixation, and where best results were obtained with thinner sections (10 to 20 microns). Going forward I would like to try this, but for the time being the tissue I have to work with is 50 microns thick and fixed in 4% PFA. With that said, could someone offer suggestions on how to achieve good staining under these conditions? Thank you for your time.
I am staining for intracellular pSTAT1 and I am repeatedly running into difficulty owing to the fact I have to permeabilise with methanol (for sufficient nuclear entry of anti-pSTAT1 antibody and to facilitate STAT dimer separation).
The methanol is wreaking havoc with certain fluorochromes/antigens and causing either no successful staining, or false/bizarre stains. The problem is that it is proving almost random as to which fluorochromes/antigens are sensitive and which are resistant, for example a certain fluorochrome will work for one particular antigen and not another, and vice versa, suggesting it's a combination of both.
As of yet, I haven't found a reliable source of information to guide the design of my experiments around this, and was wondering if anyone had any ideas or know of any resource that may help me!
Thanks in advance!
Hi all!
Can a mouse whole vertebral column be stored in 4% PFA for longer than 24 hours, but shorter than one week?
I plan to put the vertebral columns of my mice in PFA on a Friday and do sucrose the following reagents on Monday. Will this affect the quality of the extracted spinal cord later on?
Thanks in advance for your help.
Cheers!
Hi everybody,
I need a protocol and buffers to extract proteins from mouse hippocampus samples for ELISA measurement. The protein that I am going to measure is NR2B-NMDA. Samples are freeze in -80 degrees celcius.
I would like to detect motor neurons in mouse spinal cord as an alternative to violet crystal.
I need protocol for FACS analysis of Rat Sciatic nerve.
When he gives extract orally for 14 days and challenged by lps once, and performed novel object recognition. When he analysed his data, results was not dose dependent 100 and 200 mg is dose dependent but they declined in 400 mg but toxicity studies says (literature) LD may be up to 2000 mg. So what are all the possible reasons for this case.
Please share your suggestions
Thanks in advance
obviously theres a group of CFS'ERS AND ME/CFS'ERS that didn't get ill from viruses but instead became seceptable to them and re-activation of them and some were made ill by chemical exposures, now MCS and the sinus,olfactory,brain pathway, I think shows a whole other impact to the brain besides the stomach/bowel route. GWI has been reconized as TBI/PTSD, YET THERES STILL SO LITTLE POINTING TO THE SEVERITY OF BRAIN DAMAGE that turned my world upside down when I too fit into this box , I cant tell when my brain is affected by either route and no not all ME/CFS'ERS have the sinus/brain involvement witch is looking to not only be related to chronic rhinosinusitis but very possably severe Fibromyalgia. I know we are getting there but gee, kind of would like to see it happen before I croak! obviously there are some in the know out there yet it seems limited to GWI research, very thankful for that but hey some of us are not GWV'S yet suffer from the same cause "chemical exposure" there's a few new articles out on MCS , one that addresses the hearing dysfunctions, can we get more articles out there that point to chemical exposures and the tipping point (what I call it) where mast cells may suppress the immune system when they cant keep up and this allows mucosal and tissue damage and infection to acure and not only play a role in autoimmunity but probably also the B cell switching to IgE. I can relate to both GWI and Autism ,have tried to keep up with research on both.
Dear all, may I know if microglia cells produce leukotrienes in any form?
Thank you.
Looking for studies in adolescent monkeys regarding development of neuroimmune mechanisms (e.g.,microglia) or even peripheral immune mechanisms (e.g., phagocytosis, lymphocyte numbers). I'm coming up empty--any leads?
I injected a drug (subcutaneous in mice) and I noticed that there was an increase in number of microgias in the hippocampus (IBA positive cells). These microglias are more branched and non-reactive to MHC-II.
How should I interpret this increased in the number of branches and increased in the number of not activated microglia caused by a drug compared to control animals (which did not suffer intervention)? It can be interpreted as neuroprotection phenotype even if they have not suffered any inflammatory stimulus? Is it possible that microglia become more "resting" than those of the control animals that do not have inflammatory stimulus?
- How long would you expect auto-antibodies to be present in CSF in autoimmune encephalitis?
- Would there be any difference between untreated and treated (IVIG/rituximab) cases?
- Could you perform a lumbar puncture several years later in untreated patients and still be able to detect the auto-antibody (AMPAR 1/2, NMDAR, GABA-bR, VGCC, CASPR-2, VGKC)?
Thank you for your time and help!
I need to fix my animals with 4% Para to do IHC on the sections later. But I also need to determine the integrity of the BBB and I have chosen the IV HRP method. I have read that most others use Karnovsky's Fixative (2% Paraformaldehyde, 2.5% Glutaraldehyde and 0.1M Buffer). Any suggestions? What are the benefits to using the Karnovsky's Fixative?
Last year a link between a form of fetal brain damage and the mosquito-born Zika virus has been confirmed by Brazilian health authorities.
The virus, endemic in parts Africa, South America, Southeast Asia and some Pacific Islands, has until now been blamed for symptoms such as fever, mild headache, skin rashes, joint pain and conjunctivitis, or "red eye."
Initial analysis shows that the virus can be passed to a fetus and that the fetus is at greatest risk from the virus during the first three months of pregnancy.
A surge in recent months of babies born with microcephaly, or an unnaturally small brain, in Brazil's northeast, led authorities to suspect the virus may have more sinister effects than previously recorded.
Microcephalic children can suffer developmental and intellectual difficulties that limit intelligence and muscle coordination for life.
I am trying obtain primary microglial cells from adult mice. I am using magnetic bead separation and from FACS analysis my cultures are pretty pure (around 90%) but every time I plate the cells they die!
I am using DMEM/F12 + Glutamax media and coating them onto poly-L-lysine plates. There are one or two cells that are alive, and look like microglia in each field of view but the majority are small, round and live/dead stain showed they were positive for PI.
I want to try it again but I am not sure why they are dying and what I should change next time.
Any advice/help would be great appreciated!!
Thanks,
Sam
Hello everyone! I would like to find a lab host to participate to TALENTs grant. It is a ìn european grant that provide salary for 18 months, divided in 12 to spent in an Host institution and 6 to return to my base institution. I'm looking for an host laboratory situated in one of following country: Slovenia*, Croatia, Bosnia and Herzegovina, Serbia, Montenegro, Albania, Greece, Germany, France, Austria, Switzerland, Liechtenstein. The laboratory, given my CV, should ideally work in neuroinflammation / neurodegenerative diseases or I would like to learn CRISPR/CAS9 system...details in the link attached..
thanks for all the possible answers!
We want to culture hippocampal slices and we want to selectively deplete astrocytes and oligodendrocytes without affecting the neurons. We know how to deplete microglial cells and it works more or less effectively. Is there any compound (like clodronate) to deplete these cells pharmacological? We found some papers describing methods but I don't think that they are applicable in our experiments. Thanks for your answers =)!
Hi. I used mixed glial culture, then shake the primary microglia off and replate them in 96 well plate for functional assay. I saw different morphology between WT cell and gene A knockout cell. I enclosed here the CD11b staining picture of both cell types. The WT cell more look like the ramified shape, and the gene A KO cell looks more like an amoeba shape. What is the normal morphology of primary microglia from mixed glial culture? Does the gene A KO cell more like activated microglia?
Thanks!
I am trying to detect cytokines TNF and IL1B in primary rat glia culture media (high cofluency, no phenol red) after 40 min treatment with amyloid b oligomers. Unfortunately I could never reach detection limit (15 pg/ml). I could also not detect anything in lysed cultures (hypotonic buffer) and after 10x concentration of cell culture media. I would be very grateful if somebody could share their experience in ELISA-based detection of cytokines in cell culture media.
To improve data and reduce variability i would like to move away from outbred strain (Sprague Dawley) and use inbred rats. But it is nearly impossibly to work out which strain to use from the available ones: Lewis, Long Evans, Fischer, Kyoto and many more. I know, for example, that Lewis rats are used for some neuroimmunology (EAE model) but this is different to our model (effect of systemic LPS on the brain) and therefore might not be directly relevant. Do people have any experience with this type of work and inbred rats?
The process of myelinogenesis starts at birth and last until 22 years of age.
Sleep may play an important role in this process.
In demyelinising diseases such MS sleep disturbances are the rule.
Perhaps it will be interesting for this studies the myelin mutant taiep rat model
by José R. Eguibar, Instituto de Fisiología, Universidad Autónoma de Puebla. Mexico)
Neurotoxin, Cuprizone, is used for demyelination in mouse brain for de and remyelination. My inquiry is how it demyelinates?
Humoral response has a profound effect on M.S. symptoms
We are finding a significant role of neuro invasive viruses in the instigation and promotion of the Multiple Sclerosis. Is there any other scientist out there who has already conducted some research work on this subject?
I require a mRNA marker for microlgia for qPCR analysis and was wondering if IFNg would be a suitable candidate. I am looking at microglia in quail and commerical gene sequences are hard to come by but I have one for IFNg. I undertsand IFNg activates microglia so would this be a viable candidate?#
Thanks
I've been trying to culture human fetal microglia from whole human fetal brain samples. I've been using the protocol by Durafort, et al. (link attached). Of course the first time I tried the protocol it worked great, but every sample thereafter has been resulting in nearly complete cell death.
The first time I did the prep there were clearly lots of cells that stuck to the flask the next day and my total microglia yield was on target for what the authors reported. Now the cells that are there barely stick to the flask and are easily lifted just by gently moving the flask, and they look nothing like the cells that I got the first time I completed the prep.
The one difference is that the first time I used heat-inactivated FBS and since then I've been using charcoal-stripped FBS. Otherwise the media is the same (DMEM, 5% FBS, 1% pen/strep).
As I've only tried the prep a couple of times, I'm wondering if this is something with sample variability or maybe even if there is something going wrong in how the samples are being shipped to us (in media vs. PBS, in which case the cells may just be dying in the PBS). I just completed a prep yesterday and will have another look in a few days (I don't want to touch the flasks just in case any cells may get lifted).
If you happen to know of a method that works or tips or tricks that work for them I would really appreciate it! If you have any questions for me let me know. Thanks!
Macrophages are known to be an active player in MS. They are involved in active demyelination and myeline uptake, which leads towards the development of foamy macrophages with M2-like properties. But can they migrate out of the brain/lesion before they become necrotic and cause a pro-inflammatory respons (cfr. atherosclerosis)?
I am trying to prepare an anisomycin solution for injection into the brains of my rats. I am dissolving the ANI in equimolar HCl ( 1 mmol of ANI for 1mmol of HCl). The ANI dissolves easily in HCl. I then diluted the HCl a bit with saline, which was also no problem, but when I adjusted the pH of my solution with NaOH the ANI fell out of solution again.
Any suggestion?
Thanks
At present I am member of the Laboratory of Clinical Neuroimmunology in Institut de Recerca del Hospital Universitari Vall d'Hebron (Barcelona, Spain). We are concerned with Multiple Sclerosis research and now we are isolating Treg cells from human blood. I have read researchers to obtain about million cd4+cd25+cd127lo/- cells from 20 mL blood sample. After several attempts, we get an average of half a million cells from 64 mL blood sample. We use the CD4+CD25+CD127dim/- Regulatory T Cell Isolation Kit II (MACS, from Miltenyi Biotec). We use either one LD column and two MS columns or two LD columns and one MS column. Could I know which protocol do you follow? Thanks.
I am considering using a scholl analysis (using imagej software) but I was wondering if anyone has any better ideas. I only need to determine if the microglia are activated, I don't need to describe their morphology in detail.
Could you suggest any good marker and corresponding antibody you employed to mark synapses? PSD95 seems a suitable option. I am using it on primary neuron culture from mouse cortex and hippocampus, but if it would work on cerebellar cultures would be also fine.
Thanks
francesco
I'm looking for an IHC or mRNA marker that specifically identifies ramified (unactivated) microglia so I can distinguish between an inactive and active phenotype. Can anyone help please? Thanks
I'd like to perform an experiment on mice inducing EAE with whole myelin as the immunogen. The most important reference seems to be "Oct;21(4):749-57.
Myelination in rat brain: method of myelin isolation. Norton WT, Poduslo SE.J. of Neurochemistry 1973" that obviously is about rats.. Thank you in advance for your help.
Recently I checked the acsf pH before and after 95% O2/ 5% CO2 buffering and found pH value is 7.8 (7.8) and 7.6 (after) with 26mM NaHCO3 and 5% CO2. Does anyone know how to calculate the buffer ability of CO2-NaHCO3 pair? And what's the pH range of your acsf before and after buffering?
Dear all, I am Rajaram.
At present I am doing depression studies on animals. I also want to check the same in vitro on cell lines. If any cell lines are there (like BV-2 microglia) please suggest some cell lines. Which is the ideal cell line for checking the LPS-induced depressive like behavior in vitro?
Thanks in advance..
I am interested to know why C6 is the famous cell line to be chosen to co-culture with bEnd.3 (endothelial cells).
I am trying to induce passive EAE by adoptive transfer of primed T cells from WT C57BL/6 donors to WT C57BL/6 recipients. The protocol I followed was:
Day 0 - immunised donor mice with 200ug MOG35-55 s.c. + one does of 200ng pertussis toxin i.p.
D 8 - sacrifice donors and remove lymph nodes and put cells in culture with 20ug/ml MOG + 10ng/ml IL-23
Day 11 - blasting cells observed. Transferred 10 million cells i.p. per sub-lethally irradiated WT recipient (irradiated approximately 3 hours prior to cell injection). =D0 of scoring EAE.
Now it is Day 15 post cell transfer and my mice are not displaying any EAE symptoms. I see some protocols also give pertussis with the cell injection into recipients and again two days later? Or did I inject too few cells? What could the problem be?
I am searching for human microglial cell lines (best would be immortalized and easily transfectable) and I would really appreciate any hint or comment.
And neurodegeneration in mice.
Tau oligomers and aggregates have been shown to be the major target, on the basis of our neuroimmunomodulation theory of Alzheimer´s pathogenesis.
A-804598 is an antagonist for P2X7 receptors. We're trying to find out if there's an in vivo data in relation to toxicity for this particular antagonist. Please let me know if there's anything, much appreciated.
- It's an 8 weeks chronic mild stress mice model, interest being the polarization of microglia/macrophages and NLRP3 inflammasome activation in CNS. Administration route is via gavaging syringe. (updated 08/04/2014)
I still don't know what kind of injection I am going to perform (intraperitoneal or intracerebral) to obtain the best staining.´Does anyone has experience in what could be the best way of injection/timing of LPS? Thanks!
I am looking for protocols to develop a co-culture system and then introduce a virus in the culture so that the macrophage can present it to the T-cells.
We need to identify neurons in the brainstem, but cannot use NeuN made in mouse, which is the most frequently used, because our other antibodies are made in mouse, and I cannot find in the literature any experimental work with rabbit NeuN. Has anybody used any commercial one? thanks!!
I am at the annual BD nordic meeting, where the possibility of BD producing fluorescently labelled anti-mouse dopamine receptors antibodies suitable for flow cytometry has come up if there is enough interest in the scientific community. Anybody interested? Which subtypes would be the most interesting?
I am working on a connection with colic and brain development in infants.
Mice are perfused with saline and 4% PFA, followed by 4hr fixation in 4% PFA. Brains are then placed in 30% sucrose overnight, then sectioned at 30um on a freezing microtome. Sections are then placed on slides, dried and washed with PBS. I then utilized a variety of different antigen retrieval steps, blocking buffers, and antibody dilutions. The following protocols are what I've tried so far:
Wash (3x PBS)
Block for 60 min with 4% NGS in 1% BSA, 0.4% TX-100 PBS
Incubate in 1° Ab (1:1k with Anti-Iba1 from WAKO, Ab is for IHC) overnight at 4C
Wash
Peroxidase removal with 1% H2O2 for 15min
Wash
Incubate in 2° ab (Goat Anti-rabbit) in BSA/TX/PBS
Wash
ABC incubation
Wash
DAB
This protocol results in very spotty staining pattern, various regions of cortex stain, nothing in the striatum or hypothalamus. I've tried both free floating and staining on slides. Where the staining does work it works beautifully, I see complete microglia morphology and no background stain. However this only occurs in ~5% of any tissue section (not 5% of samples, but 5% of a tissue slice).
So I tried a variety of different antigen retrieval steps including
50 mM TBS + 0.05% Tween 20, pH 9.0 for 20 min at 99°C
10mM TE + 0.05% Tween 20, pH 9.0 for 20 min at 99°C
10mM NA citrate buffer + 0.05% Tween 20, pH 6.0 for 30 min at 80°C
Tissue was then blocked with 10% Fetal Bovine Serum, 3% BSA, tx-PBS for 60min then incubated ON in Anti-Iba1 (1:2.5k, WAKO)
Then I followed the above protocol
This resulted in mixed but complete staining.
The tissue incubated in TBS had very weak, but uniform Iba staining (only cell bodies stained, no processes).
Tissue incubated in TE showed a stronger uniform Iba staining but again only cell bodies stained.
Tissue incubated in SSC showed the best staining pattern, I now see some processes, but its still far from how surveying microglia should appear, plus I now have massive background staining.
So I tried the SSC antigen retrieval method again, this time I blocked with 4% NGS, BSA/Tx/PBS, and followed the above protocol. This results in the worst staining I've yet to see, massive background only cell bodies stain.
Any thoughts, tips, tricks, etc for staining Iba-1 in mouse brain are very welcome!
The protocol is working very well in Polyethylene glycol embedded tissue. All of the sections are labelled on slides. We have to use 6u sections so that we can get sufficient cell counts with BrdU/NeuN. However, we are trying to process these same sections for numbers of activated and ramified microglial numbers in the hippocampus. I am having difficulties determining how to analyze the morphology of the ramified microglial cells, as their cell bodies are very small and their processes are extending into other sections of tissue. Any ideas?